The blood brain barrier (BBB) dynamically regulates the movement of proteins from the circulation to the central nervous system (CNS). In general, the BBB excludes peptides and proteins in the periphery from substantial entry into the CNS. The ability to treat neurological disorders with protein and peptide drugs is largely hindered by this effect. Therefore, by learning how some microbes successfully cross the BBB we may gain much needed insight for the development of drug delivery into the CNS. Tight junctions in cerebral microvascular endothelial cells form the BBB. Pathogens may cross the BBB through paracellular, transcellular, and/or Trojan horse mechanisms. Microbial proteins appear to be the major determinants of the ability of neurotropic pathogens to cross the BBB. Various bacterial proteins that contribute to BBB penetration by transcytosis have been identified in gram positive and gram-negative bacteria. In contrast, little is known about the spirochetal proteins involved in crossing of the BBB. Many spirochetes, including Treponema pallidum and the several Borrelia species that cause Lyme disease and relapsing fever cross the BBB. The focus of our laboratory is to understand the pathogenesis of neurological infection with Lyme disease and relapsing fever borrelias, referred to as neuroborreliosis. In mice infected with the relapsing fever spirochete Borrelia turicatae the site of entry into the CNS is the cerebral microcirculation. Relapsing fever borrelias are best known for antigenic variation due to sequential expression of variable major lipoproteins (VMP's) divided in two families, Variable Large Proteins (Vlp's) and Variable Small Proteins (Vsp's). During persistent infection of antibody-deficient mice with isogenic serotypes of B. turicatae we found that spirochetes expressing Vspl (Btl) caused 10 times more CNS infection and inflammation than spirochetes expressing Vsp2 (Bt2) even though Bt2 spirochetes were 10 times more abundant in blood. More recently we found that recombinant Borrelia burgdorferi expressing Vspl bind to human brain microvascular endothelial cells (BMEC) more than if Vsp2 is expressed, and that Vspl binds to BMEC cell membranes. These results implicate Vspl as the first known spirochetal "neuroinvasin". Although the localization of Btl in the CNS is leptomeningeal, there is widespread cerebral microgliosis in infected mice suggesting that spirochetal products may diffuse into the brain parenchyma. The goal of this R21 application is to study further the interaction between Vspl and the cerebral microcirculation. Our main assumption is that Vspl crosses brain microvascular endothelial cell barriers. The specific hypotheses for this project are that (i) Vspl can enter the CNS from the circulation, and (ii) Specific polymorphisms in the distal variable loops of Vspl explain its high affinity for the cerebral microcirculation. We propose to study these hypotheses with the following specific aims: 1. Determine whether Vspl crosses the BBB in vivo. 2. Investigate whether Vspl enters BMEC. 3. Determine whether Vspl crosses BMEC barriers. 4. Identify the structural features that mediate the interaction of Vspl with BMEC. [unreadable] [unreadable]